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At least 289 records · Page 16Linked to original sources
PubMed1998

A novel mutation (F227L) arises in the reverse transcriptase of human immunodeficiency virus type 1 on dose-escalating treatment of HIV type 1-infected cell cultures with the nonnucleoside reverse transcriptase inhibitor thiocarboxanilide UC-781.

Treatment of wild-type human immunodeficiency virus [HIV-1(IIIB)]-infected cell cultures with the thiocarboxanilide UC-781 under low selective pressure (i.e., 0.01 microg/ml) resulted in the emergence of V106A RT mutant virus. On increasing drug concentrations (stepwise up to 30 microg/ml) the virus retained the V106A RT mutation but acquired the novel F227L mutation in the RT genome in addition to the L100I, K1O1I, and Y181C mutations. This multiple-mutant virus proved highly resistant to virtually all nonnucleoside RT inhibitors (NNRTIs) (e.g., nevirapine, delavirdine, and loviride), but retained full sensitivity to nucleoside analogs such as AZT, ddI, (-)FTC, and 3TC. The F227 amino acid is highly conserved in HIV-1 strains and forms part of the NNRTI-binding pocket. Our model suggests a hydrophobic interaction between F227 and the chloro atom of UC-781.

Anilides↗
PubMed2006

Multi-drug resistant HIV-1 reverse transcriptase genotype in children treated with dual nucleoside reverse transcriptase inhibitors (NRTIs).

BACKGROUND: Multi-drug resistant HIV mutants have been reported after prolonged dual antiretroviral therapy. OBJECTIVE: To evaluate the prevalence and resistance pattern in HIV-infected children treated with dual NRTIs. MATERIAL AND METHOD: Records of HIV-infected children treated with dual NRTIs at Srinagarind Hospital, Khon Kaen University, Thailand, were reviewed for baseline data and their consensually-stored plasma were checked for the occurrence of HIV mutants by genotyping. RESULTS: Fifty-seven HIV-infected children were treated with dual NRTI regimens (27 males; 30 females). The median age and median CD4+ T-lymphocyte at genotypic testing were 83.5 months and 10.9%, respectively. The median duration of ARV therapy was 22 months. More than half the children (42) were on zidovudine and didanosine. A set of three or more nucleoside analog mutations (NAMs), conferring multi-dideoxynucleoside resistance, was found in 60% of the cases. CONCLUSION: High percentages of NAMs were found in HIV-infected children previously on dual ARV therapy for long periods. Genotypic testing was helpful in designing the second antiretroviral regimen.

Anti-HIV Agents↗
PubMed2003

Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: comparison of real-time reverse transcription-PCR vs. relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products.

The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.

Algorithms↗
PubMed1999

Similarities and differences in the RNase H activities of human immunodeficiency virus type 1 reverse transcriptase and Moloney murine leukemia virus reverse transcriptase.

Retroviral revXerse transcriptases (RTs) have an associated RNase H activity that can cleave RNA-DNA duplexes with considerable precision. We believe that the structure of the RNA-DNA duplexes in the context of RT determines the specificity of RNase H cleavage. To test this idea, we treated three related groups of synthetic RNA-DNA hybrids with either Moloney murine leukemia virus (MLV) RT or human immunodeficiency virus type 1 (HIV-1) RT. All of the hybrids were prepared using the same 81-base RNA template. The first series of RNase H substrates was prepared with complementary DNA oligonucleotides of different lengths, ranging from 6 to 20 nucleotides, all of which shared a common 5' end and were successively shorter at their 3' ends. The second series of oligonucleotides had a common 3' end but shorter 5' ends. The DNA oligonucleotides in the third series were all 20 bases long but had non-complementary stretches at either the 5' end, 3' end, or both ends. Several themes have emerged from the experiments with these RNA-DNA duplexes. (1) Both HIV-1 RT and MLV RT cleave fairly efficiently if the duplex region is at least eight bases long, but not if it is shorter. (2) Although, under the conditions we have used, both enzymes require the substrate to have a region of RNA-DNA duplex, both MLV RT and HIV-1 RT can cleave RNA outside the region that is part of the RNA-DNA duplex. (3) The polymerase domain of HIV-1 RT uses certain mismatched segments of RNA-DNA to position the enzyme for RNase H cleavage, whereas the polymerase domain of MLV RT does not use the same mismatched segments to define the position for RNase H cleavage. (4) For HIV-1 RT, a mismatched region near the RNase H domain can interfere with RNase H cleavage; cleavage is usually (but not always) more efficient if the mismatched segment is deleted. These results are discussed in regard to the structure of HIV-1 RT and the differences between HIV-1 RT and MLV RT.

Amino Acid Sequence↗
PubMed2005

Evaluation of an oligonucleotide ligation assay for detection of mutations in HIV-1 subtype C individuals who have high level resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.

The oligonucleotide ligation assay (OLA) has been proposed as an affordable alternative to sequence-based HIV-1 drug resistance testing in resource poor settings. The aim was to evaluate OLA for detecting mutations K103N, Y181C, K65R, Q151M, M184V and T215Y/F in subtype C. Forty-four subtype C and 8 subtype B HIV-1 positive individuals were analysed using the ViroSeqtrade mark HIV-1 genotyping assay (Applied Biosystems, Foster City, CA). A one-step RT-PCR and nested PCR were performed using subtype B specific primers from the OLA kit (NIH AIDS Research and Reference Reagent Program). Seventy-eight subtype C sequences were used to design subtype C specific primers. Ligation and detection steps were followed according to OLA kit protocol. For codons, K103N, Y181C, K65R, Q151M, M184V and T215Y/F, four or more mismatches compared to the probe or mismatches less than four bases from the ligation site were not tolerated. Results revealed accurate identification of mutations in 2/10, 4/9 3/9, 6/7, 2/7 and 6/7 VQA samples and 5/20, 4/17 0/20, 18/24, 5/24 and 13/24 subtype C positive individuals, respectively. It was concluded that the probes and primers in the NIH reference kit would need modification to optimize detection of mutations in subtype C individuals.

Anti-HIV Agents↗